Nucleotide-resolution mapping of regulatory elements via allelic readout of tiled base editing
CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.
Authors
- Madelynn N. Whittaker (ORCID: https://orcid.org/0000-0002-3382-1134)
- Elise Sylvander (ORCID: https://orcid.org/0009-0005-9981-2942)
- Davide Seruggia (ORCID: https://orcid.org/0000-0001-5014-0499)
- Manfred Lehner (ORCID: https://orcid.org/0000-0002-5776-4218)
- Ana P. Kutschat (ORCID: https://orcid.org/0000-0002-6360-873X)
- Basheer Becerra (ORCID: https://orcid.org/0000-0003-4403-7959)
- Sandra Wittibschlager
- Christina S. Horstmann
- M M Starrs
- Eric Che
- Anzhelika Tauber
- Zain M. Patel
- Luca Pinello
- Michael I. Love
- Martin Jankowiak
- Sophie Müller
- Ting Wu
- Benjamin P. Kleinstiver
- Daniel E. Bauer
- Justin Delano
Institutions
- Broad Institute (US)
- University of North Carolina at Chapel Hill (US)
- Boston Children's Hospital (US)
- Harvard University (US)
- Austrian Academy of Sciences (AT)
- Massachusetts General Hospital (US)
- Christian Doppler Laboratory for Thermoelectricity (AT)
- Harvard College Observatory (US)
- Dana-Farber Cancer Institute (US)
- St Anna Children's Hospital (AT)
- CeMM Research Center for Molecular Medicine (AT)
- Dana-Farber/Harvard Cancer Center (US)
- Center for Cancer Research (US)
- Harvard Stem Cell Institute (US)
- St. Anna Children's Cancer Research Institute
- Community College of Rhode Island (US)
Publication Details
- Journal
- Nature Communications
- Published
- 2026-03-03
- DOI
- https://doi.org/10.1038/s41467-026-69918-8
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Österreichische Nationalstiftung für Forschung, Technologie und Entwicklung
- Austrian Science Fund
- Christian Doppler Forschungsgesellschaft